Efficacy of non-surgical interventions for promoting improved functional outcomes following acute compartment syndrome: A systematic review

Background Acute compartment syndrome (ACS) is a devastating complication which develops following a traumatic extremity injury that results in increased pressure within osteofascial compartments, thereby leading to ischemia, muscle and nerve necrosis, and creates a life-threatening condition if left untreated. Fasciotomy is the only available standard surgical intervention for ACS. Following fasciotomy the affected extremity is plagued by prolonged impairments in function. As such, an unmet clinical need exists for adjunct, non-surgical therapies which can facilitate accelerated functional recovery following ACS. Thus, the purpose of this systematic review was to examine the state of the literature for non-surgical interventions that aim to improve muscle contractile functional recovery of the affected limb following ACS. Methods English language manuscripts which evaluated non-surgical interventions for ACS, namely those which evaluated the function of the affected extremity, were identified as per PRISMA protocols via searches within three databases from inception to February 2022. Qualitative narrative data synthesis was performed including: study characteristics, type of interventions, quality, and outcomes. Risk of bias (RoB) was assessed using the Systematic Review Centre for Laboratory Animal Experimentation’s (SYRCLE) RoB tool and reported level of evidence for each article. Results Upon review of all initially identified reports, 29 studies were found to be eligible and included. 23 distinct non-surgical interventions were found to facilitate improved muscle contractile function following ACS. Out of 29 studies, 15 studies which evaluated chemical and biological interventions, showed large effect sizes for muscle function improvement. Conclusions This systematic review demonstrated that the majority of identified non-surgical interventions facilitated an improvement in muscle contractile function following pathological conditions of ACS.


Introduction
The majority of combat-related injuries within the military population are in the lower extremities [1][2][3]. Acute compartment syndrome (ACS) within the extremities is a devastating condition that can develop in conjunction with long bone fractures, crush injury, gunshot wounds, burns, contusions, over-exertion, or prolonged limb compressions [4,5]. ACS is also seen in civilian populations who are involved in fire arm assaults, motorcycle crash, and pedestrian struck by automobile injuries [6]. ACS occurs when the pressure within a closed compartment space surpasses a critical threshold (perfusion pressure of 30 mmHg or less) and compromises circulation/perfusion subsequently leading to ischemia [7][8][9][10]. At the biochemical level, reactive oxygen species (ROS) generated within the ischemic tissues activate a neutrophil response via arachidonic acid inflammatory pathways [11] that ultimately leads to intracellular swelling/edema, vascular and nerve damage, and deep muscle pain at the affected tissue site. Moreover, this inflammatory response further accelerates the release of cytotoxic enzymes and excess ROS whereby an additional cascade of cellular damage is triggered. Under these conditions, skeletal muscle, specifically Type II muscle fibers, are susceptible to severe damage leading to myonecrosis and a loss of function with a prolonged/delayed recovery [12]. As such, while loss of function presents a threat of limb loss, release of cellular contents from myonecrosis in conjunction with complement activation, and pro-inflammatory cytokines accelerate the systemic inflammation which can lead to remote organ damage [13][14][15] and contribute to multi-organ failure, or even death if left untreated [11,[16][17][18][19][20]. Therefore, it is essential to relieve pressure, reduce ischemia, and restore blood circulation to salvage ischemic tissues shortly after diagnosis to limit the primary and secondary impacts of the condition.
Fasciotomy is the only standard surgical intervention available to treat ACS. Surgical treatment of ACS by fasciotomy restores perfusion within the injured compartment with immediate relief; however, in-so-doing, this procedure further contributes to an additional reperfusion injury due to the release of pro-inflammatory immune cells and by-products of muscle tissue damage [7] and a further increase in oxidative stress. In other words, reperfusion injury is multifactorial and causes extensive tissue destruction involving generation of ROS, calcium overload, capillary leakage/endothelial dysfunction, the opening of the mitochondrial permeability transition pore, thus impairing ATP production, activation of inflammatory and pro-thrombogenic cascades, mechanically and metabolically induced cytoskeletal damage. Ultimately, ACS and the associated ischemia reperfusion (I/R) injury elicits a deleterious impact on the muscle contractile functionality of the affected limb. There exists an opportunity for the development of adjunct, non-surgical therapeutics which aim to facilitate improved functional outcomes by increasing the rate of healing and reducing the overall recovery time for those afflicted with ACS.
Given the lack of clinically available non-surgical treatments for ACS, it is imperative to better understand the existing literature, across the translational research continuum, to this end. Therefore, the purpose of this study was to perform a systematic review of the ACS literature to identify all non-surgical therapeutics which aim to promote improved functional outcomes that have been investigated as a means of providing a reference for subsequent use in the development of next generation technologies. While the initial literature search conducted to this end resulted in no articles recapitulating trauma-induced ACS with outcomes on muscle function in extremities, evaluation of articles which examined interventions on tissue I/R injuries were used as a means for identifying therapeutic candidates which might address the primary aspects of the pathophysiology of ACS in extremity trauma.

Search strategy
A systematic review of the literature was conducted in accordance with the Preferred Reporting Items for Systematic review and Meta-Analysis (PRISMA) protocols [21]. This systematic review was not registered and a review protocol paper was not submitted. A comprehensive search of several databases (PubMed, Web of Science and Embase) was conducted by one author (N.B.J) from inception to Feb 3 rd , 2022 using the following key word strategy: ("Trauma" OR "penetrating" OR "injury" OR "wound") AND ("extremity" OR "limb" OR "arm" OR "leg") AND ("acute compartment syndrome" OR "compartment syndrome" OR "crush syndrome" OR "intracompartmental pressures" AND "edema" OR "edema" OR "ischemia" OR "ischemia" OR "reperfusion") AND ("therapy" OR "drug" OR "intervention" OR "treatment" OR "pharmacology" OR "medicine" OR "therapeutic" OR "small molecule" OR "biologic") AND ("muscle function" OR "muscle strength" OR "neuromuscular strength").

Selection criteria
Covidence software was used for primary screening and data extraction. The articles were reviewed in an un-blinded, standardized method. Two independent reviewers (NBJ and JMM) reviewed the title and abstract of all studies for eligibility and subsequently screened the full texts of those included. Discrepancies were resolved by a third reviewer (SMG). To be included in the analyses, each article was screened for all of the following criteria: 1) original studies containing primary data about non-surgical interventions for traumatic ACS of extremities, 2) utilization of a skeletal muscle functional outcome measure, 3) availability of the full text of the article, 4) reported sample size, measured variance as standard deviation (SD) of the mean or standard error of the mean (SEM) 5) reporting of the type of intervention, and 6) duration of treatment. The following exclusion criteria were applied: 1) non-traumatic, chronic, exertional, and/or pathological forms of compartment syndrome, vascular ACS and abdominal ACS, 2) studies including patients below 18 years of age, 3) biomechanical studies, ex-vivo/invitro studies, 4) non-primary data reports (e.g., technical reports, reviews, commentaries, conference abstracts and case-reports), 5) studies in languages other than English, 6) studies with no functional analysis of the affected musculature, and 7) non-traumatic healthy patients study.

Preclinical studies
The following details from included studies were extracted by two independent reviewers (NBJ and JMM): 1) publication year and the name of the first author, 2) the characteristics of the in-vivo model including sample size, species, sex, weight, and age, 3) anesthesia methods 4) symptoms such as edema, inflammation, and loss of function, 5) information related to treatment group(s), including type of therapeutic agent (e.g., biological or chemical), therapeutic dosage, method of administration, duration of treatment, and therapeutic agent alone or combined with another material and the same information of control group, 6) types of outcome measures evaluated, such as inflammation, edema, and muscle strength/function, and 7) group sizes, mean value and SD or SEM of outcomes. If outcomes were performed at different time points, only data from the final test was extracted for use herein. If the experimental group of animals received various doses of the drug therapy, all the data of various doses of the drug was extracted for use herein. Muscle function data was extracted from each experimental and control group of every study. Data was extracted from published figures (for unreported actual data values) using Web Plot Digitizer, version 4.5 (https://automeris.io/ WebPlotDigitizer). The effect size with 95% CI was for the muscle functional outcome measure was calculated for each individual study.

Study quality and risk of bias of the studies
Two independent reviewers (NBJ and JMM) utilized the Systematic Review Centre for Laboratory Animal Experimentation's (SYRCLE) risk of bias (RoB) tool to assess the risk of bias [22] via the following criteria: 1) peer reviewed publication, 2) control of temperature and lighting for animal housing, 3) random allocation of animals to treatment or control groups, 4) timing of disease induction before/after randomization (ischemia/trauma), 5) random selection of animals for outcome assessment, 6) blinded assessment of outcome, 7) use of anesthetic without significant effects on the skeletal muscle injury/force production, 8) animal model (aged, young, sex, weight), 9) sample size calculation, 10) free of selective outcome reporting (i.e., if all the pre-specified primary and secondary outcomes are reported), 11) compliance with animal welfare regulations, and 12) statement of potential conflict of interests. Study quality is rated as yes scored 1, and studies rated as no or unsure scored 0. Each study quality was given overall quality of evidence scores. Lower total scores indicate a higher risk of bias.

Data analysis and synthesis
A qualitative narrative data synthesis of included studies was conducted. Study characteristics, type of interventions, quality, and outcomes were reported according to a standard format and similarities and differences compared across studies.

Study selection
Database searches yielded 608 titles with potential relevance (Fig 1). After excluding 62 duplicate studies, 546 studies were screened for titles and abstracts, of which 466 studies did not meet the inclusion criteria leaving 80 studies eligible for full text review (Fig 1). After reviewing the full text, an additional 51 studies were excluded due to the following reasons: no therapeutic investigated (n = 15), no full text available (n = 15), irrelevant study design/studies (e.g. prophylactic or other forms of compartments syndrome that are not traumatic ACS of the extremities) (n = 8), no evaluation of muscle function (n = 6), article not in English (n = 3), case report (n = 3), or a review article (n = 1). Subsequently, a total of 29 studies met the criteria for inclusion (See Table 1).
The experimental endpoints varied considerably across studies ( Table 1). The majority of studies terminated their experiments within 28 hours [24-26, 28-32, 35-38, 46-48, 50]. Only one study out of 29 terminated after 40 days [51]. Three studies terminated the experiments after 28 days [23,34,45] and another study terminated at 21 days [44]. Two studies terminated the experiments after 16 days [41,42]. Two studies terminated the experiments at 14 days [33] and one of the studies terminated at two different time points, seven and 14 days [39]. One study terminated the experiments after 7 days [40]. One study terminated the experiments after five days [27]. One study terminated the experiments after two days [49].
Inflammation and edema. Out of the 29 studies, 11 reported inflammatory results ( Table 2). Out of the 11 studies which reported inflammatory results, one study reported the inhibition of inflammation based on histological analysis of inflammatory cells such as, neutrophils, lymphocytes, and macrophages [23], one study reported the drug effects on inflammation based on microscopic observations (no details on how these observations were made are reported) [24], two studies reported inhibition of inflammation based on histology analysis of neutrophil infiltration alone [25,36], and of the remaining six studies, two reported results based on CD68 immunoreactivity indicative for macrophage infiltration [33,48], two studies from the same group [26,27] and one other study [29] employed myeloperoxidase activity as a marker of skeletal muscle neutrophil infiltration to study inflammation, and the remaining study analyzed stained tissue for cellular inflammation with no specifics on the type of inflammatory cells [35], and one study analyzed inflammasome pathway genes (NLRP3, caspase 1, IL-1β, and IL-18) to study the drug effects on inflammation [45] ( Table 2).

Muscle isometric torque
In general, functional outcome measures improved after treatment in 22 of the included studies. All studies that explored chemical interventions showed an improvement in maximum Table 3. Methodological qualities of each study and their scores.

Summary of the overall results
No clinical studies qualified for inclusion within this systematic review. Within the preclinical literature, the efficacy of the non-surgical interventions for ACS with respect to edema, inflammation, and muscle-contractile function was assessed in 29 studies. Although edema and inflammation are the key players in ACS pathology, not all of the included studies investigated the effects of interventions on these symptoms. 22 studies showed improvement in muscle contractile function after the treatments. The evidence available from the present study revealed that 12 chemical interventions (i.e., SNAC, dexamethasone, L-carnitine, hypertonic saline, EPC-K1, L-NMMA, taurine, ATP/MgCl2, TCA, DCA, DMSO, and mannitol) while seven biological interventions (i.e., FWB, Lin-BMCs, CYR61, bone marrow-derived mesenchymal stem cells (MSCs), LPS/IFN-γ polarized mouse bone marrow macrophages, mMPs, and exosomes) and three combinatorial interventions (i.e. chemical and biological co-therapies: SOD/DMSO/mannitol and Sodium percarbonate and catalase) were found to facilitate improvement in muscle contractile function. Likewise, One physical intervention (i.e. warmer reperfusion liquid (maintained at 34˚C-36˚C) was shown to mediate an improvement in muscle isometric torque after injury. Effect size calculations revealed that out of all the studies, the study which tested NC-exosomes expressing cPWWP2A repaired I/R injury by inhibiting Rb1-mediated NLRP3 inflammasome through the cPWWP2A/Rb1/AMPKα2/NLRP3 signaling pathway showed the largest effect on muscle function improvement. The remaining studies which showed large effect sizes on muscle function improvement are: SNAC, dexamethasone, hypertonic saline, pravastatin, low dose of sodium percarbonate, SOD (at five hours), DMSO

Limitations of the included studies
This review provides useful information with respect to the possibility of using above identified agents as non-surgical therapies to elicit improved functional outcomes following ACS. However, the articles included within this systematic review have the following limitations: 1) None of the studies provided information on sample size calculations. The adequate sample size is required for scientific rigor and appropriate interpretation of findings. 2) Only one It is important to study anesthetic effects on muscle injury. There are speculations that inhalant anesthetics may directly involve the contractile proteins actin or myosin. Thus, having control animals to study anesthesia effects alone is recommended to account for these variables. 5) Only 11 studies included the statement "disclosure of potential conflict" out of 29 studies. Declaring conflicts of interest is critical for maintaining the integrity of unbiased professional assessment of the publications. Previously, the inclusion of this statement was largely neglected in reputed journals, but presently, it is necessary to report conflicts of interest statements before acceptance for publication. 6) The majority of included studies, except for three, reported random allocation of animals to treatment or control groups. It is well known that failure to randomize will lead to the overestimation of treatment benefits of interventions across outcome measures. 7) Out of 29 studies, six did not specify the timing of ischemia/trauma induction before/after randomization, making the analysis difficult. As discussed earlier, the randomization of animals plays an important role in outcome assessment.
Further, only five studies adopted female animals, and it cannot be ignored that both genders will be developing ACS due to trauma with a different pattern of sensitivity towards trauma.
Healing patterns may also differ in these genders due to differences in the type of sexual hormones. Another limitation is that none of the studies used a crushed/trauma model to create ischemia for testing intervention efficacy on muscle function. As discussed previously, immediately after ACS is diagnosed, the fascia should be cut open within 3-6 hrs of the injury to prevent irreversible damage. This point should be considered when designing the experiments. Another limitation is that three of the included studies terminated the study within 1 or 2 hrs, which is before the onset of ACS. Early termination of studies made it difficult to derive/ understand results that are meaningful to apply for treating trauma-related ACS. Although these models represent the pathophysiological mechanism that causes ACS with increased pressure and ischemia leading to tissue damage, clearly, all of the models reviewed here are not representative of the exact pathophysiology of ACS present in the trauma patient population, where severe destruction of soft tissue with/without fractures, localized reductions in regional blood flow to traumatized tissue and trauma induced inflammatory milieu are seen. As discussed earlier, high-quality animal studies are crucial for the translation of animal data into clinical studies [52]. Therefore, we suggest that while designing experiments/analyzing results, authors should try to avoid the limitations listed above and follow standard guidelines for animal studies to improve the accuracy of the experimental data [53].

Implications
As previously discussed, fasciotomy is the current standard of care for ACS and the only clinical option for immediate relief of the increased intra-compartmental pressure. Part of the sequelae of ACS and its subsequent release via fasciotomy is a reperfusion injury which carries with it a multi-week timeline over which muscle function is slowly recovered. Given the prevalence of ACS in military trauma, this recovery timeline is a significant burden to our wounded Service members and ultimately the readiness of the joint Forces. As such identifying and gaining knowledge of the effective treatments for reperfusion injury is paramount to designing adjunct, non-surgical treatment strategies to fasciotomy for ACS. Thus, given the lack of bonafide ACS studies in the literature, studies focused on preventing or ameliorating the pathophysiology of I/R injury were used to gain insights into how various readily available interventions might help reduce ACS symptoms and improve muscle function. The majority of the studies evaluated here terminated the experiments at/after >4 hrs except for three studies [30,31,47]. Biological interventions used individually are the most effective in protecting the tissue from I/R damage and improved muscle contractile function. These interventions tested muscle contractile function after a prolonged time (7 to 40 days after intervention) compared to other treatments, which analyzed this parameter within 24 hrs. Despite the relevance of studies that investigated the intervention effects on I/R damage within 24 hours of injury, they are limited in understanding the effects of interventions that may prolong the ischemic time that tissue can resist before the tissue damage occurs. An increase in the length of the ischemic period will lead to an increase in cell death with irreversible damage followed by loss of structural integrity of the affected tissue. Therefore, specifically targeted therapies are necessary to activate cell survival programs to overcome the pathologic events associated with ACS.
From the reviewed literature, it is apparent that inflammation plays a significant role in creating ACS conditions [54]. Compounds that possess anti-inflammatory properties may show improved efficacy in reducing ACS, importantly when improving muscle contractile function in the affected limb. A majority of the included studies showed improvement in muscle function whereas, only 10 included studies reported/analyzed effects on inflammation. Out of 10 studies, eight studies showed inhibitory effects on inflammation after treatments. Similarly, edema is the result of acute inflammation from an injury [54]. Edema results in an increased tissue pressure, metabolic insults due to tissue necrosis and increased infiltration of inflammatory cells, inducing defective endogenous muscle recovery mechanisms. Thus, the development of therapeutic strategies is required to minimize the pathological inflammatory processes and direct towards tissue formation. Among the included studies, only a few aimed to analyze intervention effects on edema. Currently, this review was able to show that the majority of interventions tested in the included studies rescued the muscle from pathological conditions of ACS with an improvement in muscle contractile function. The agents discussed in this review may have the potential to rescue the muscle from pathological conditions of ACS developed by trauma.
From this paper, we have observed a lack of therapies which directly target skeletal muscle regeneration as a means to accelerate functional recovery after ACS. Given the critical role that myofiber damage and necrosis plays in the pathology of ACS, it seems that such interventions represent an underexplored therapeutic option which warrants further investigation as a means to accelerate functional recovery of the afflicted. To this end interventions (e.g. small molecules or direct cellular therapies) which might promote an expanded pool of activated satellite cells in the affected compartment would be of particular interest either as a monotherapy or in conjunction with a number of compounds targeting inflammation, oxidative stress, or nitric oxide metabolism as reviewed herein.

Conclusions
Based on the findings presented herein, both chemical and biological interventions may improve muscle-contractile function following I/R within an ACS conditions. However, caution should be exercised in the interpretation of the data as variability in experimental designs, analysis measures, and limited analysis of edema and inflammation limits our understanding of how these interventions compare to each other. In addition, there are paucity of studies available on the use of extremity trauma-mediated ACS models which utilized muscle contractile force as an (primary) outcome measure. Therefore, further work is warranted to evaluate the efficacy of non-surgical interventions on functional outcomes in ACS that is caused by extremity trauma. Further research in this direction will help wounded soldiers who develop ACS to have shorter recovery times and restoration of full muscle function.